Microparticle protection of therapeutic agents

ABSTRACT

The present invention is directed to the use of microparticles to protect the pharmaceutical effectiveness of a pharmaceutically active agent. According to one embodiment, a pharmaceutically acceptable suspension is provided that comprises microparticles and a pharmaceutically active agent. This pharmaceutically acceptable suspension is then exposed to a component or condition that is incompatible with the pharmaceutically active agent, such that the microparticles provide a pharmaceutical effectiveness that is greater than it would have been in the absence of the microparticles. Preferably, the microparticles result in a pharmaceutical effectiveness of the pharmaceutically active agent that is at least 10% greater than the pharmaceutical effectiveness of the pharmaceutically active agent would have been in the absence of the microparticles. Polymer microparticles, such as polystyrene microparticles, are one preferred class of microparticles. The microparticles preferably range from 0.01 to 100 microns in largest dimension, more preferably 0.1 to 10 microns in largest dimension. The microparticles are preferably provided in an amount of 0.1 to 1 wt % within the suspension. Agents comprising polynucleotides, including cells, plasmids and viral vectors, are a preferred class of pharmaceutically active agent. Other embodiments on the invention are directed to pharmaceutically acceptable suspensions, medical devices for parenteral injection, and methods of treatment.

STATEMENT OF RELATED APPLICATIONS

This is a divisional application and claims the benefit of priority toco-pending U.S. patent application Ser. No. 09/845,080, filed Apr. 27,2001, and entitled “Microparticle Protection of Therapeutic Agents,which is related to U.S. Ser. No. 09/429,178 filed Oct. 28, 1999, nowU.S. Pat. No. 6,638,259. This application is also related to U.S. Ser.No. 09/503,586 filed Feb. 14, 2000, now U.S. Pat. No. 6,663,606. Each ofthese applications is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to the protection of therapeutic agents and moreparticularly to novel techniques and compositions for the protection oftherapeutic agents using microparticles.

BACKGROUND OF THE INVENTION

As noted in related U.S. Ser. Nos. 09/429,178 and 09/503,586, deviceshaving metallic and polymeric components are used extensively in themedical field. In many cases, such medical devices are used for deliveryof a solution or suspension containing a pharmaceutically active agent,and the pharmaceutically active agent comes into contact with themetallic or polymeric component during the course of its delivery.Metallic materials used in such devices include stainless steel andnickel-titanium superelastic alloys (e.g., nitinol). Polymericcomponents used in such devices include polycarbonate, polyimide,acrylonitrile/butadiene/styrene resins (ABS), poly ether ether ketone(PEEK), epoxy-based adhesives (such as FDA2 or FDA23) and nylon (such asnylon 6,6). The inventors in related U.S. Ser. Nos. 09/429,178 and09/503,586 have found, however, that despite their reputation as beingsubstantially inert, such materials can be incompatible to varyingdegrees with certain pharmaceutically active agents.

The present invention provides a simple and unexpected way of overcomingthe above and other incompatibilities.

SUMMARY OF THE INVENTION

According to an embodiment of the present invention, a method isprovided in which microparticles are used to protect the pharmaceuticaleffectiveness of a pharmaceutically active agent. The method comprises:(a) providing a pharmaceutically acceptable suspension comprising thepharmaceutically active agent and microparticles; and (b) exposing thepharmaceutically acceptable suspension to a component or condition thatis incompatible with the pharmaceutically active agent, such that themicroparticles result in a pharmaceutical effectiveness of thepharmaceutically active agent that is greater than it would have been inthe absence of the microparticles. Preferably, the microparticles resultin a pharmaceutical effectiveness of the pharmaceutically active agentthat is at least 10% greater than the pharmaceutical effectiveness ofthe pharmaceutically active agent would have been in the absence of themicroparticles.

Components incompatible with the pharmaceutically active agent includemetals (such as certain stainless steel and nickel-titanium alloys),polymers (such as certain poly ether ether ketones, polyimides, epoxies,nylons, acrylonitrile/butadiene/styrene polymers and polycarbonates) andglass.

Conditions incompatible with the pharmaceutically active agent includefreeze-thaw transformations.

The microparticles preferably range from 0.01 to 100 microns in largestdimension, more preferably 0.1 to 10 microns in largest dimension. Themicroparticles are preferably provided in an amount ranging from 0.1 to1 wt % within the suspension. Polymer microparticles, such aspolystyrene microparticles, are one preferred class of microparticles.

According to another embodiment of the invention, a method of treatmentis provided. The method comprises: (a) providing a pharmaceuticallyacceptable suspension comprising a pharmaceutically active agent andmicroparticles; (b) providing a medical device having a component thatis incompatible with the pharmaceutically active agent; and (c)parenterally injecting the pharmaceutically active agent into a patientvia the device while at the same time removing the microparticles fromthe pharmaceutically acceptable suspension. Preferred devices includeparenteral injection devices, such as vascular catheters and syringes.

According to another embodiment of the invention, a device forparenteral injection is provided that comprises: (a) a pharmaceuticallyacceptable suspension comprising a pharmaceutically active agent andmicroparticles; (b) a device component that contacts the suspension andis incompatible with the pharmaceutically active agent; and (c) aseparator that acts to remove the microparticles from thepharmaceutically acceptable suspension prior to parenteral injection.

According to another embodiment of the invention, a pharmaceuticallyacceptable suspension is provided. The suspension comprises: (a) apharmaceutically active agent; and (b) microparticles that prevent asubstantial reduction in pharmaceutical effectiveness of thepharmaceutically active agent upon being exposed to a material orcondition that is incompatible with the pharmaceutically active agent.

One advantage associated with the present invention is that the efficacyof pharmaceutically active agents can be protected in a simple manner.

Another advantage of the present invention is that a pharmaceuticallyactive agent can be stored in a storage container or delivered from amedical device that contains materials that would otherwise result in asubstantial reduction in the pharmaceutical effectiveness of thepharmaceutically active agent.

Another advantage of the present invention is that a pharmaceuticallyactive agent can be stored under conditions that would otherwise resultin a substantial reduction in the pharmaceutical effectiveness of thepharmaceutically active agent.

Yet another advantage of the present invention is that an agent (i.e.,microparticles) can be provided to protect the efficacy of thepharmaceutically active agent, but need not be introduced into a patientat the time of administration.

These and other embodiments and advantages of the present invention willbecome readily apparent to those of ordinary skill in the art uponreview of the detailed description and claims to follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph illustrating viral activity of an adenoviralsolution, with and without microparticles, according to an embodiment ofthe present invention.

DETAILED DESCRIPTION

As a preliminary matter, it is noted that “pharmaceutical article”, asdefined herein, means any article that comes into contact with apharmaceutically active material.

By “pharmaceutical effectiveness” or “pharmaceutical efficacy” is meantany desired pharmaceutical result. As a specific example, thepharmaceutical effectiveness of a virus can be measured by the abilityof that virus to infect cells. As another example, the pharmaceuticaleffectiveness of a protein can be measured by its activity within anELISA assay.

Pharmaceutical effectiveness is said to be “substantially reduced”, is“substantially lower” or is said to undergo a “substantial reduction”when it is reduced, for example, by at least 5%, more commonly 10%, 20%,30%, 40%, 50% or more. An “incompatible component” is a component thatcauses a substantial reduction in pharmaceutical effectiveness uponcontacting a pharmaceutically active material. A condition is an“incompatible condition” that, when encountered by a pharmaceuticallyactive material, results in a substantial reduction in pharmaceuticaleffectiveness.

Pharmaceutical effectiveness is said to be “substantially increased”, is“substantially higher” or is said to undergo a “substantial increase”when it is increased, for example, by at least 5%, more commonly 10%,20%, 30%, 40%, 50% or more.

At present, many pharmaceutical articles, including various medicaldevices, are known in which solutions or dispersions of pharmaceuticallyactive agents come into contact with materials prior to delivery to thebody. However, as seen below in the Examples, and as further shown inrelated U.S. Ser. Nos. 09/429,178 and 09/503,586, where certainpharmaceutically active agents contact substrates comprising certainmaterials, their pharmaceutical effectiveness is substantially reduced.

For instance, it has been found that where viral particles contactcertain metallic materials, such as certain stainless steel and/ornickel-titanium alloys (e.g., nitinol), or they contact certainpolymeric materials, such as certain poly ether ether ketones (PEEK),polyimides, epoxies, nylons, acrylonitrile/butadiene/styrene resins(ABS) and/or polycarbonates, viral transfection may be substantiallyreduced. This is surprising, since it is normally assumed that suchmetallic and polymeric materials are substantially inert and henceunlikely to adversely affect pharmaceutically active agents.

The present invention overcomes these and other difficulties through theuse of microparticles that substantially protect the pharmaceuticaleffectiveness of pharmaceutically active agents upon encounteringmaterials or conditions that are incompatible with the active agents.

It is well within the skill of those of ordinary skill in the art todetermine which materials, in addition to those specifically listedabove, are incompatible with a given pharmaceutically active agent.Possible mechanisms for a substantial reduction in pharmaceuticaleffectiveness include inactivation (e.g., through denaturation,precipitation, damage, and so forth) and adsorption of thepharmaceutically active agent. It is also well within the skill of thoseof ordinary skill in the art to determine which conditions areincompatible with a given pharmaceutically active agent.

The present invention utilizes microparticles to substantially protectthe pharmaceutical effectiveness of pharmaceutically active agents uponcontacting incompatible materials or encountering incompatibleconditions. The microparticles and the pharmaceutically active agent arepreferably provided in a suspension. As a result of the presence of themicroparticles in the suspension, the pharmaceutically active agent issubstantially protected upon contact with the incompatible materials orexposure to the incompatible conditions.

By “substantially protected” is meant that effectiveness of thepharmaceutically active agent is substantially greater in the presenceof the microparticles, relative to the effectiveness of thepharmaceutically active agent in the absence of the microparticles.

The microparticles of the present invention can be made of essentiallyany material that is effective to achieve protection of thepharmaceutically active agent, without resulting in a pharmaceuticallyunacceptable outcome (e.g., unacceptable levels of toxicity). Thesematerials include materials that are not incompatible with thepharmaceutically active agent, including certain metals (e.g., gold,titanium and platinum), ceramics/glasses (e.g., quartz), polymers, andcombinations of the same.

Polymers appropriate for the practice of the invention may becrosslinked or uncrosslinked, linear or branched, natural or synthetic,thermoplastic or thermosetting, or biostable, biodegradable,bioabsorbable or dissolvable.

Exemplary polymers include the following polymers and copolymers:polycarboxylic acid polymers and copolymers including polyacrylic acids(e.g., acrylic latex dispersions and various polyacrylic acid productssuch as HYDROPLUS, available from Boston Scientific Corporation, NatickMass. and described in U.S. Pat. No. 5,091,205, the disclosure of whichis hereby incorporated herein by reference, and HYDROPASS, alsoavailable from Boston Scientific Corporation); acetal polymers andcopolymers; acrylate and methacrylate polymers and copolymers;cellulosic polymers and copolymers, including cellulose acetates,cellulose nitrates, cellulose propionates, cellulose acetate butyrates,cellophanes, rayons, rayon triacetates, and cellulose ethers such ascarboxymethyl celluloses and hydoxyalkyl celluloses; polyoxymethylenepolymers and copolymers; polyimide polymers and copolymers such aspolyether block imides, polybismaleinimides, polyamidimides,polyesterimides, and polyetherimides; polysulfone polymers andcopolymers including polyarylsulfones and polyethersulfones; polyamidepolymers and copolymers including nylon 6,6, polycaprolactams andpolyacrylamides; resins including alkyd resins, phenylic resins, urearesins, melamine resins, epoxy resins, allyl resins and epoxide resins;polycarbonates; polyacrylonitriles; polyvinylpyrrolidones (cross-linkedand otherwise); anhydride polymers and copolymers including maleicanhydride polymers; polymers and copolymers of vinyl monomers includingpolyvinyl alcohols, polyvinyl halides such as polyvinyl chlorides,ethylene-vinylacetate copolymers (EVA), polyvinylidene chlorides,polyvinyl ethers such as polyvinyl methyl ethers, polystyrenes,styrene-butadiene copolymers, acrylonitrile-styrene copolymers,acrylonitrile-butadiene-styrene copolymers, styrene-butadiene-styrenecopolymers and styrene-isobutylene-styrene copolymers, polyvinylketones, polyvinylcarbazoles, and polyvinyl esters such as polyvinylacetates; polybenzimidazoles; ionomers; polyalkyl oxide polymers andcopolymers including polyethylene oxides (PEO); glycosaminoglycans;polyesters including polyethylene terephthalates and aliphaticpolyesters such as polymers and copolymers of lactide (which includeslactic acid as well as d-,l- and meso lactide), epsilon-caprolactone,glycolide (including glycolic acid), hydroxybutyrate, hydroxyvalerate,para-dioxanone, trimethylene carbonate (and its alkyl derivatives),1,4-dioxepan-2-one, 1,5-dioxepan-2-one, and6,6-dimethyl-1,4-dioxan-2-one (a copolymer of polylactic acid andpolycaprolactone is one specific example); polyether polymers andcopolymers including polyarylethers such as polyphenylene ethers,polyether ketones, polyether ether ketones; polyphenylene sulfides;polyisocyanates (e.g., U.S. Pat. No. 5,091,205 describes medical devicescoated with one or more polyisocyanates such that the devices becomeinstantly lubricious when exposed to body fluids); polyolefin polymersand copolymers, including polyalkylenes such as polypropylenes,polyethylenes (low and high density, low and high molecular weight),polybutylenes (such as polybut-1-ene and polyisobutylene),poly-4-methyl-pen-1-enes, ethylene-alpha-olefin copolymers,ethylene-methyl methacrylate copolymers and ethylene-vinyl acetatecopolymers; fluorinated polymers and copolymers, includingpolytetrafluoroethylenes (PTFE),poly(tetrafluoroethylene-co-hexafluoropropene) (FEP), modifiedethylene-tetrafluoroethylene copolymers (ETFE), and polyvinylidenefluorides (PVDF); silicone polymers and copolymers; polyurethanes (e.g.,BAYHYDROL polyurethane dispersions); p-xylylene polymers;polyiminocarbonates; copoly(ether-esters) such as polyethyleneoxide-polylactic acid copolymers; polyphosphazines; polyalkyleneoxalates; polyoxaamides and polyoxaesters (including those containingamines and/or amido groups); polyorthoesters; biopolymers, such aspolypeptides, proteins, polysaccharides and fatty acids (and estersthereof), including fibrin, fibrinogen, collagen, elastin, chitosan,gelatin, starch, glycosaminoglycans such as hyaluronic acid; as well asblends and copolymers of the same.

Those of ordinary skill in the art will be able to determine whichpolymers are most appropriate for a given pharmaceutically activematerial with relative ease using, for example, techniques like thoseused in the Examples.

Latex beads represent one preferred class of polymer microparticles thatare useful in connection with the present invention. Natural latexes aswell as synthetic latexes (e.g., latexes formed by emulsionpolymerization from polystyrene, styrene-butadiene copolymers, acrylatepolymers, polyvinyl acetate, and so forth) are preferred, withpolystyrene latexes being more preferred.

The term “microparticle” as used herein refers to small particlesranging in largest dimension from 0.01 to 1000 microns, preferably 0.01to 100 microns, more preferably 0.1 to 10 microns, and even morepreferably about 1 micron. While substantially spherical particles(including both spheres and beads) are preferred, particles of anyshape, including rod-shaped particles and irregularly shaped particles,are contemplated.

The microparticles and pharmaceutically active agents are providedwithin any physiologically acceptable liquid medium known in the art,including physiological saline, phosphate buffered saline, and solutionscontaining trehalose, sucrose, glycerol, tris(hydroxymethyl)aminomethanebuffer and/or MgCl₂. Additional adjuvants known in the art are alsocontemplated.

Preferred amounts of the microparticles range from 0.01 to 10 wt %within the suspension, more preferably 0.1 to 1 wt %.

Preferred amounts of the pharmaceutically active agents aretherapeutically effective amounts; such amounts are well within theability of those of ordinary skill in the art to determine.

“Pharmaceutically active agents”, “pharmaceutically active materials”,“therapeutic agents”, “drugs” and other related terms are usedinterchangeably herein and include genetic therapeutic agents,non-genetic therapeutic agents and cells. Pharmaceutically active agentsuseful in accordance with the present invention may be used singly or incombination.

Therapeutic agents include cells of human origin (autologous orallogeneic), including stem cells, or from an animal source(xenogeneic), which can be genetically engineered if desired to deliverproteins of interest. Cell types include bone marrow stromal cells,endothelial progenitor cells, myogenic cells including cardiomyogeniccells such as procardiomyocytes, cardiomyocytes, myoblasts such asskeletomyoblasts, fibroblasts, stem cells (e.g., mesenchymal,hematopoietic, neuronal and so forth), pluripotent stem cells,macrophage, satellite cells and so forth. Cells appropriate for thepractice of the present invention also include biopsy samples for directuse (e.g., whole bone marrow) or fractions thereof (e.g., bone marrowstroma, bone marrow fractionation for separation of leukocytes). Whereappropriate, media can be formulated as needed to maintain cell functionand viability.

Therapeutic agents also include both polymeric (e.g., proteins, enzymes)and non-polymeric (e.g., small molecule therapeutics) agents and includeCa-channel blockers, serotonin pathway modulators, cyclic nucleotidepathway agents, catecholamine modulators, endothelin receptorantagonists, nitric oxide donors/releasing molecules, anesthetic agents,ACE inhibitors, ATII-receptor antagonists, platelet adhesion inhibitors,platelet aggregation inhibitors, coagulation pathway modulators,cyclooxygenase pathway inhibitors, natural and syntheticcorticosteroids, lipoxygenase pathway inhibitors, leukotriene receptorantagonists, antagonists of E- and P-selectins, inhibitors of VCAM-1 andICAM-1 interactions, prostaglandins and analogs thereof, macrophageactivation preventers, HMG-CoA reductase inhibitors, fish oils andomega-3-fatty acids, free-radical scavengers/antioxidants, agentsaffecting various growth factors (including FGF pathway agents, PDGFreceptor antagonists, IGF pathway agents, TGF-β pathway agents, EGFpathway agents, TNF-α pathway agents, Thromboxane A2 [TXA2] pathwaymodulators, and protein tyrosine kinase inhibitors), MMP pathwayinhibitors, cell motility inhibitors, anti-inflammatory agents,antiproliferative/antineoplastic agents, matrix deposition/organizationpathway inhibitors, endothelialization facilitators, blood rheologymodulators, as well as integrins, chemokines, cytokines and growthfactors.

Therapeutic agents also include genetic therapeutic agents and proteins,such as ribozymes, anti-sense polynucelotides and polynucleotides codingfor a specific product (including recombinant nucleic acids) such asgenomic DNA, cDNA, or RNA. The polynucleotide can be provided in “naked”form or in connection with vector systems that enhances uptake andexpression of polynucleotides. These can include DNA compacting agents(such as histones), non-infectious vectors (such as plasmids, lipids,liposomes, cationic polymers and cationic lipids) and viral vectors suchas viruses and virus-like particles (i.e., synthetic particles made toact like viruses). The vector may further have attached peptidetargeting sequences, antisense nucleic acids, and DNA chimeras whichinclude gene sequences encoding for ferry proteins such as membranetranslocating sequences (“MTS”) and herpes simplex virus-1 (“VP22”).

Further therapeutic agents include:

-   -   Anti-sense DNA and RNA    -   tRNA or rRNA to replace defective or deficient endogenous        molecules    -   Gene delivery agents, which may be either endogenously or        exogenously controlled. Examples of endogenous control include        promoters that are sensitive to a physiological signal such as        hypoxia or glucose elevation. Exogenous control systems involve        gene expression controlled by administering a small molecule        drug. Examples include tetracycline, doxycycline, ecdysone and        its analogs, RU486, chemical dimerizers such as rapamycin and        its analogs, etc.    -   Angiogenic molecules including:        -   growth factors: such as acidic and basic fibroblast growth            factors, vascular endothelial growth factor, epidermal            growth factor, transforming growth factor alpha and beta,            platelet-derived endothelial growth factor, platelet-derived            growth factor, platelet derived endothelial cell growth            factor, tumor necrosis factor α, hepatocyte growth factor,            insulin like growth factor, placental growth factor; PR39,            angiogenin, prostaglandin E1 and E2, interleukin 8,            angiopoietins (I, II. III, IV, etc), androgens, proliferin,            granulocyte colony stimulating factor, estrogens        -   transcription factors: such as Hif1a, Del1,        -   protein kinases: such as Akt    -   Cytotoxic factors or cell cycle inhibitors, including CD        inhibitors: such as p53, thymidine kinase (“TK”) and other        agents useful for interfering with cell proliferation    -   The family of bone morphogenic proteins (“BMP's”): including        BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 (Vgr-1), BMP-7 (OP-1), BMP-8,        BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, and        BMP-16. Currently preferred BMP's are any of BMP-2, BMP-3,        BMP-4, BMP-5, BMP-6 and BMP-7. These dimeric proteins can be        provided as homodimers, heterodimers, or combinations thereof,        alone or together with other molecules. Alternatively or, in        addition, molecules capable of inducing an upstream or        downstream effect of a BMP can be provided. Such molecules        include any of the “hedgehog” proteins, or the DNA's encoding        them.    -   Cell survival molecules: including Akt, insulin-like growth        factor 1, NF-kB decoys, 1-kB,    -   Other therapeutic agents: including Madh6, Smad6, Apo A-1,    -   Small molecule activators or inhibitors of the genes described        above including decoys.    -   Vectors and gene transfer agents including:        -   Viral vectors: such as adenoviruses, gutted adenoviruses,            adeno-associated virus, retroviruses, alpha virus (Semliki            Forest, Sindbis, etc.), lentiviruses, herpes simplex virus,            ex vivo modified cells (i.e., stem cells, fibroblasts,            myoblasts, satellite cells, pericytes, cardiomyocytes,            sketetal myocytes, macrophage, etc.), replication competent            viruses (ONYX-015, etc.), and hybrid vectors.        -   Nonviral vectors: artificial chromosomes and            mini-chromosomes, plasmid DNA vectors (pCOR), cationic            polymers (polyethyleneimine, polyethyleneimine (PEI) graft            copolymers such as polyether-PEI and polyethylene oxide-PEI,            neutral polymers PVP, SP 1017 (SUPRATEK), lipids or            lipoplexes, nanoparticles and microparticles with and            without targeting sequences such as the protein transduction            domain (PTD).

A “polynucleotide” is a nucleic acid molecule polymer, such as DNA, RNAand their analogs, having as few as 3 nucleotides, and can include bothdouble- and single-stranded sequences. A “protein” is a polymer of asfew as two (dimer) amino acid residues.

Preferably, the pharmaceutically active material is a cell orpolynucleotide, more preferably a cell or polynucleotide that is presentin the form of a plasmid or that is present in conjunction with virus orvirus-like particles. Specific examples of preferred cells includecardiomyocytes, skeletal myoblasts, endothelial cells, and stem cells.Specific examples of preferred virus or virus-like particles includeadenovirus, paroviruses such as adeno-associated virus, lentivirus,retrovirus, alpha-virus, papilloma virus, murine leukemia virus, SemlikiForest virus, and so forth.

To form the suspensions of the present invention, the microparticles andthe pharmaceutically active agent are commingled in a liquid medium byessentially any known means, including stirring, shaking, and so forth.

After commingling, the suspension containing the microparticles and thepharmaceutically active agent can be stored in any manner known in theart. In one preferred embodiment, the suspension is stored in an ampoule(i.e., a sealed container, typically glass or plastic, which contains asterile solution for parenteral injection) until the time ofadministration.

The use of the microparticle suspensions of the present invention with(a) manufacturing articles, including fermentors, glassware,plasticware, probes and tubing, (b) other storage and transportarticles, including storage vessels, transport vessels, stoppers, lidsand septums, and (c) analytical articles, including needles, pipettetips, cell culture apparatus and analytical equipment, is alsocontemplated.

Where the therapeutic agent is in solution or is substantially smallerthan the microparticles used, separation of the therapeutic agent andmicroparticles can be carried out with relative ease. For example, themicroparticles can be separated from the therapeutic agent by strainingthe microparticles from the suspension, for example, by passing thesuspension through a filter of an appropriate pore size (or, as anotherexample, through a screen of appropriate mesh) prior to administrationto a patient. Preferred patients are mammalian patients, more preferablyhuman patients.

For instance, as set forth in the Examples below, the addition of1-micron polystyrene latex beads to a virus suspension has been shown toessentially retain virus activity when exposed to an incompatiblematerial. In these Examples, the beads are on the order of 10 times thesize of the viral particles. As a result of this large sizedifferential, the polymeric beads can be easily excluded from aninjectate by placing a size exclusion mesh at the distal end of adelivery lumen, permitting injection of the viral particles withoutinjection of the polymeric beads. Similarly, where loss of activity isrelated to storage and where the medical device is not incompatible withthe therapeutic agent, the beads can be separated prior to associationof the therapeutic agent with the medical device.

Alternatively, by selecting biocompatible microparticles, themicroparticles can be administered to a patient along with thetherapeutic agent. Referring again to the Examples below, the latexpolystyrene beads are tissue compatible and may be injected along withthe viral particles to the patient. Injection of the beads is believedto enhance both cellular gene transfer and in vivo stability.

In many preferred embodiments of the present invention, themicroparticle suspensions are administered to patients via drug-deliverymedical devices and accessories. Contemplated medical devices arenumerous. For example, the medical devices contemplated for use inconnection with the present invention can be those used for systemictreatment or those used for local treatment of a tissue or organ.Non-limiting examples include tumors; organs including but not limitedto the heart, lung, brain, liver, kidney, bladder, urethra and ureters,eye, intestines, stomach, pancreas, ovary, and prostate; skeletalmuscle; smooth muscle; breast; cartilage; and bone.

Essentially any medical device for parenteral injection (i.e.,administration by a route other than the alimentary canal, includingsubcutaneous, intramuscular, intravenous, intravascular, intraorbital,intracapsular, intraspinal and intrasternal administration) iscontemplated for use in connection with the present invention.

Preferred medical devices include catheters, including endoluminalcatheters such as needle injection catheters (e.g., for endocardial,epicardial, and pericardial agent administration), balloon catheters,diagnostic catheters and perfusion catheters, conventional needlesyringes, hypodermic needles, intravenous injection devices, biopsyneedles and devices, tissue ablation devices, aspirating needles,stents, and so forth. Specific examples of devices for drug delivery tothe heart include, for example, those found in the following patents andpatent applications: U.S. Pat. No. 5,450,846, U.S. Pat. No. 5,840,059,U.S. Pat. No. 5,878,751, U.S. Pat. No. 5,551,427, U.S. Pat. No.5,931,834, U.S. Pat. No. 5,925,012, U.S. Pat. No. 5,925,033, U.S. Pat.No. 5,538,504, WO 99/39624, WO 99/44656, WO 99/21510, WO 99/29251, EP A99-06 0895752, and EP A 99-01 0888750, each of which is incorporatedherein by reference.

In some cases, the microparticles are provided because the entiremedical device is composed of an incompatible material. In other cases,only a portion of the medical device is composed of such incompatiblematerials.

The present invention is particularly useful in connection with viraldelivery from percutaneous transcatheter devices.

In other preferred embodiments of the present invention, themicroparticles are used to protect the effectiveness of pharmaceuticallyactive agents under conditions related to storage. For example, themicroparticles can be used to protect the activity of virus suspensionsduring storage and during freeze-thaw.

Below are Examples directed to a specific embodiment for carrying outthe present invention. The Examples are offered for illustrativepurposes only, and are not intended to limit the scope of the presentinvention in any way.

The Examples are directed to the protection of adenoviral vectors.Adenoviral vectors are a highly efficient way to transfer geneticmaterial. Systemic delivery of adenoviral vectors is not preferable dueto non-target organ infection. On the other hand, administration ofadenovirus gene therapies using a medical device platform provides ameans of site-specific delivery with minimal systemic leakage. However,vector instability persists as a major limitation of this strategy.Specifically, as noted in U.S. Ser. No. 09/429,178 and U.S. Ser. No.09/503,586, medical devices have been shown to negatively impactadenoviral activity. See also Marshall et al., Molecular Therapy 2000,1(5): 423-429.

EXAMPLE 1

A CMV-LacZ adenovirus (i.e., an adenoviral vector driven by acytomegalovirus promoter and β-galactosidase reporter gene based on theLacZ enzyme) is used as a stock virus in this example.

An adenoviral solution having an Ad_(CMV-LacZ) titer of 1×10⁹ functionalunits per milliliter (fu/ml) in PBS (−/−) was prepared.

At the same time a suspension is prepared by combining the following:(a) 90 vol % adenoviral solution at a titer appropriate to give a finaltiter of 1×10⁹ fu/ml and (b) 10 vol % Fluoresbrite™ YG Microspheres fromPolysciences Inc., which contains 1.0-micron fluorescent polystyrenebeads at a concentration of 2.5% solids in water. This combinationresults in a suspension that contains a final adenovirus titer of 1×10fu/ml as well as 0.25% solids as microspheres (due to the 10:1 dilutionof the bead solution).

Boston Scientific Corporation Stiletto™ direct injection catheters,which have a proximal portion formed from heat-treated stainless steeland a distal portion formed from a nitinol hypotube, were filled withthe each of the above and incubated for 30 minutes at 37° C.

Catheter effluents were then collected and titered on HeLa cells (humanepidermoid carcinoma cells). For this purpose, HeLa cells were firstplated out in well plates at 70% confluency the day before theexperiment. Prior to contacting the HeLa cells, the viral solution wasdiluted appropriately in infection media (Dulbecco's Modified Eagle'sMedium+2% Fetal Bovine Serum) to achieve a result of 100 to 1000infected cells per well. The diluted virus was added to the HeLa cellsin the wells and incubated at 37° C. for 1 hour. 5 mls of DMEM+10% FBSwere then added to each well, followed by incubation for 24-30 hours at37° C. After aspiration of the media, the cells were fixed in 0.5%glutaraldehyde in PBS (phosphate buffered saline) for 10 minutes. Thecells were washed twice in PBS and stained using an X-gal stainingsolution overnight at 37° C. (X-gal is5-bromo-4-chloro-3-indolyl-β-D-galactoside, which is hydrolyzed byβ-galactosidase to form a blue product). Blue cells (i.e.,galactosidase-positive cells) were counted the next day to determine thetiter.

Viral activity for both the adenoviral solution and the adenoviral/beadsuspension was measured as a function of beta-galactosidase-positivecells and residual activity was calculated as a percentage of controlsincubated in polypropylene Eppendorf tubes (used here as astandard/control) under the same conditions. The results for both theadenoviral solution and the adenoviral/bead suspension are presented inFIG. 1. As can be seen, incubation of the adenovirus solution within theStiletto™ catheter leads to an essentially complete loss of virusactivity. In contrast, the addition of 1 micron polystyrene latex beadsto the adenovirus solution results in an essentially complete retentionof virus activity.

EXAMPLE 2

Efficacy of adenovirus delivery from a standard needle was performed ina mouse model. An adenoviral solution and an adenoviral suspensioncontaining beads (10 vol % Fluoresbrite™ YG Microspheres), each havingan Ad_(CMV-LacZ) titer of 10⁹fu/50 μl, were prepared. Mice wereanesthetized and a left thoracotomy was used to expose the heart. Directepicardial injections (1×50 μl) were made into the left ventricle.Animals were sacrificed 7 days following the procedure. Whole heartswere retrieved and assayed by spectroscopic absorption at 420 nm toquantify the total amount of beta-galactosidase expressed. Absorptiondata are given below on the basis of both sample size (150 μl) and mgprotein (determined using a standard protein assay). These data indicatethat beta-galactosidase expression is higher for the injectionscontaining beads. 420 nm absorption/150 μl β-gal (A420/mg protein)Adenovirus 55.49 (51.74; 59.23) 4.0 (3.3; 4.6) Adenovirus 93.03 (110.26;75.79) 8.1 (9.0; 7.1) plus beads

Although various embodiments are specifically illustrated and describedherein, it will be appreciated that modifications and variations of thepresent invention are covered by the above teachings and are within thepurview of the appended claims without departing from the spirit andintended scope of the invention.

1. A method of treatment comprising: providing a pharmaceuticallyacceptable suspension comprising a pharmaceutically active agent andmicroparticles; providing a medical device having a component that isincompatible with said pharmaceutically active agent; and parenterallyinjecting said pharmaceutically active agent into a patient from saiddevice while at the same time removing said microparticles from saidpharmaceutically acceptable suspension.
 2. The method of claim 1,wherein said microparticles are polymer microparticles.
 3. The method ofclaim 1, wherein said microparticles are polystyrene microparticles. 4.The method of claim 1, wherein the microparticles range from 0.1 to 10microns in largest dimension.
 5. The method of claim 1, wherein thepharmaceutically active agent comprises a polynucleotide.
 6. The methodof claim 5, wherein the polynucleotide is provided within a cell, aplasmid or a viral vector.
 7. The method of claim 1, wherein said deviceis a parenteral injection device selected from a vascular catheter and asyringe.
 8. A pharmaceutically acceptable suspension comprising: apharmaceutically active agent; and microparticles, wherein saidmicroparticles are provided to prevent a substantial reduction inpharmaceutical effectiveness of said pharmaceutically active agent uponbeing exposed to a material or condition that is incompatible with saidpharmaceutically active agent.
 9. The pharmaceutically acceptablesuspension of claim 8, wherein said microparticles are polymermicroparticles.
 10. The pharmaceutically acceptable suspension of claim8, wherein said microparticles are polystyrene microparticles.
 11. Thepharmaceutically acceptable suspension of claim 8, wherein themicroparticles range from 0.1 to 10 microns in largest dimension. 12.The pharmaceutically acceptable suspension of claim 8, wherein thepharmaceutically active agent comprises a polynucleotide.
 13. Thepharmaceutically acceptable suspension of claim 12, wherein thepolynucleotide is provided within a cell, a plasmid or a viral vector.14. An ampoule containing the pharmaceutically acceptable suspension ofclaim
 8. 15. A device for parenteral injection comprising: apharmaceutically acceptable suspension comprising a pharmaceuticallyactive agent and microparticles; a device component that contacts saidsuspension and is incompatible with said pharmaceutically active agent;and a separator, said separator acting to remove said microparticlesfrom said pharmaceutically acceptable suspension prior to parenteralinjection.
 16. The device of claim 15, wherein said microparticles arepolymer microparticles.
 17. The device of claim 15, wherein themicroparticles range from 0.1 to 10 microns in largest dimension. 18.The device of claim 15, wherein the pharmaceutically active agentcomprises a polynucleotide.
 19. The device of claim 15, wherein saiddevice is a parenteral injection device selected from a vascularcatheter and a syringe.